Polyethylene glycol aldehydes

ABSTRACT

Polyethylene glycol aldehyde compounds are provided. Methods of making and using such compounds, as well as chemical intermediates are also provided.

[0001] This application claims the benefit of the U.S. ProvisionalApplication 60/398,196 filed on Jul. 24, 2002.

FIELD OF THE INVENTION

[0002] The present invention relates to polyethylene glycol aldehydes,and to related methods of making and using such derivatives, such as inthe pegylation of polypeptides and other biomolecules.

BACKGROUND OF THE INVENTION

[0003] Polyethylene glycol (“PEG”) is a linear or branched, neutralpolyether, available in a variety of molecular weights. The structure ofPEG is HO—(CH₂—CH₂—O)_(n)—H, where n indicates the number of repeats ofthe ethylene oxide unit in the PEG.

[0004] PEG and PEG derivatives have been employed to modify a variety ofbiomolecules. When attached to such molecules, PEG increases theirsolubility and increases their size, but has little effect on desirableproperties.

[0005] Advantageously, PEG conjugated biomolecules may exhibit increasedretention and delayed metabolism in the body.

[0006] A variety of PEG derivatives has been developed for suchapplications. Such PEG derivatives are described, for example, in U.S.Pat. Nos. 5,252,714; 5,672,662; 5,959,265; 5,990,237; and 6,340,742.

[0007] Two general approaches have been used for the functionalizationof PEG: (1) changing the terminal hydroxyl group, through a series ofreactions, to a more active functional group and/or (2) reaction of thePEG under controlled conditions with difunctional compounds so that oneof its functional groups reacts with the PEG polymer and the otherremains active. In most cases, several steps must be conducted toachieve the desired PEG derivatives. The desired PEG derivatives areoften produced in low yields and require a complicated purificationprocess to isolate. In addition, PEG derivatives may show nonspecificbinding to the biomolecules of interest, which can result in multiplePEGs attached to a single biomolecule and/or PEG attachment at theactive site. Multiple PEG attachments may cause difficulty inpurification of the pegylated biomolecule. Multiple PEG attachments,and/or pegylation at the active site, can also lead to decreasedactivity of the biomolecule.

[0008] It would, therefore, be advantageous to provide improved PEGderivatives suitable for conjugation with a variety of other molecules,including polypeptides and other biomolecules containing an α-aminogroup. There remains a need to provide PEG derivatives that can beproduced in high yield and purity, and that can be conjugated to providebiomolecules having improved performance characteristics.

[0009] These and other objects of the present invention are described ingreater detail below.

SUMMARY OF THE INVENTION

[0010] The compounds of the invention are aldehyde derivatives ofpolyethylene glycol, having the general formula (I):

R₁—(CH₂CH₂O)_(n)—CH₂CH₂—X—Y—NH—(CH₂)_(p)—CHO  (I)

[0011] wherein R₁ is a capping group, X is O or NH, Y is selected fromthe group consisting of

[0012] Z is a side chain of an amino acid, m is from 1 to 17, n is from10 to 10,000, and p is from 1 to 3.

[0013] The present invention also provides a compound of formula (II):

[0014] wherein R₁, m, n, and p are defined as above.

[0015] Another preferred embodiment of the present invention provides abifunctional polyethylene glycol aldehyde compound of formula (VIII):

[0016] wherein m, n, and p are is defined as above.

[0017] The present invention also provides intermediate compounds offormula (IX):

R₁—(CH₂—CH₂—O)_(n)—CH₂—CH₂—X—Y—NH—(CH₂)_(p)—CH—(OCH₂—CH₃)₂  (IX)

[0018] wherein R₁, X, Y, Z, m, n, and p are defined as above.

[0019] The present invention further provides intermediate compounds offormula (X):

[0020] wherein R₁, m, n, and p are defined as above.

[0021] Also provided is an intermediate compound of formula (XI):

[0022] wherein each m, n, and p is the same or different and is definedas above.

[0023] The present invention further provides a method of making apolyethylene glycol aldehyde comprising hydrolyzing a compound offormula (IX):

R₁—(CH₂—CH₂—O)_(n)—CH₂CH₂—X—Y—NH—(CH₂)_(p)—CH—(OCH₂—CH₃)₂  (IX)

[0024] to produce a polyethylene glycol aldehyde of formula (I):

R₁—(CH₂CH₂O)_(n)—CH₂CH₂—X—Y—NH—(CH₂)_(p)—CHO  (I)

[0025] wherein R₁, X, Y, Z, m, n, and p are defined as above.

[0026] The present invention also provides a method of making apolyethylene glycol aldehyde comprising hydrolyzing a compound offormula (X):

[0027] to produce a polyethylene glycol aldehyde of formula (II):

[0028] wherein R₁, m, n, and p are defined as above.

[0029] The present invention provides a method of making a polyethyleneglycol aldehyde comprising hydrolyzing a compound of formula (XVII):

[0030] to produce a polyethylene glycol aldehyde of formula (VIII):

[0031] wherein m, n, and p are defined as above.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention provides a variety of compounds andchemical intermediates and methods which may be used in connection withthe pegylation of polypeptides and other biomolecules. The presentinvention provides a new chemical structure for polyethylene glycolaldehydes.

[0033] The compounds of the invention are aldehyde derivatives ofpolyethylene glycol, having the general formula (I):

R₁—(CH₂CH₂O)_(n)—CH₂CH₂—X—Y—NH—(CH₂)_(p)—CHO  (I)

[0034] wherein R₁ is a capping group, X is O or NH, Y is selected fromthe group consisting of

[0035] Z is a side chain of an amino acid, m is from 1 to 17, n is from10 to 10,000, and p is from 1 to 3.

[0036] As used herein the R₁ “capping group” is any suitable chemicalgroup which, depending upon preference, is generally unreactive orgenerally reactive with other chemical moieties. The terminal aldehydegroup of the above formula permits ready covalent attachment to achemical moiety of interest, for example, to the α-amino group of apolypeptide. The R₁ capping group is selected to permit or preventbifunctionality, e.g., covalent attachment to a second chemical moietyof interest.

[0037] In the case that the capping group is generally unreactive withother chemical moieties R₁ is relatively inert. If R₁ is relativelyinert, then the structure of the resulting polyethylene glycol aldehydeis monofunctional and therefore covalently bonds with only one chemicalmoiety of interest. Suitable generally unreactive R₁ capping groupsinclude: hydrogen, hydroxyl, lower alkyl, lower alkoxy, lowercycloalkyl, lower alkenyl, lower cycloalkenyl, aryl, and heteroaryl.

[0038] As used herein, the term “lower alkyl”, means a substituted orunsubstituted, straight-chain or branched-chain alkyl group containingfrom 1 to 7, preferably from 1 to 4, carbon atoms, such as methyl,ethyl, n-propyl, isopropyl, n-butyl, sec.butyl, tert.butyl, n-pentyl,n-hexyl, n-heptyl and the like. The lower alkyl is optionallysubstituted with one or more groups independently selected from halogen,lower alkyl, lower alkoxy, lower cycloalkyl, lower alkenyl, lowercycloalkenyl, aryl, and heteroaryl.

[0039] The term “lower alkoxy” means a lower alkyl group as definedearlier which is bonded via an oxygen atom, with examples of loweralkoxy groups being methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,sec.butoxy, tert.butoxy, n-pentoxy and the like. The lower alkoxy isoptionally substituted with one or more groups independently selectedfrom halogen, lower alkyl, lower alkoxy, lower cycloalkyl, loweralkenyl, lower cycloalkenyl, aryl, and heteroaryl.

[0040] The term “lower cycloalkyl” means a substituted or unsubstitutedcycloalkyl group containing from 3 to 7, preferably from 4 to 6, carbonatoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl orcycloheptyl. The lower cycloalkyl is optionally substituted with one ormore groups independently selected from halogen, lower alkyl, loweralkoxy, lower cycloalkyl, lower alkenyl, lower cycloalkenyl, aryl, andheteroaryl.

[0041] As used herein, the term “lower alkenyl” means a substituted orunsubstituted, straight-chain or branched-chain alkenyl group containingfrom 2 to 7, preferably from 2 to 5, carbon atoms, e.g., ethenyl,butenyl, pentenyl, hexenyl and the like. The lower alkenyl is optionallysubstituted with one or more groups independently selected from halogen,lower alkyl, lower alkoxy, lower cycloalkyl, lower alkenyl, lowercycloalkenyl, aryl, and heteroaryl.

[0042] The term “lower cycloalkenyl” means a substituted orunsubstituted, cycloalkenyl group containing from 4 to 7 carbon atoms,e.g., cyclobutenyl, cyclopentenyl, cyclohexenyl and the like. The lowercycloalkenyl is optionally substituted with one or more groupsindependently selected from halogen, lower alkyl, lower alkoxy, lowercycloalkyl, lower alkenyl, lower cycloalkenyl, aryl, and heteroaryl.

[0043] The term “aryl” means a phenyl or naphthyl group which isunsubstituted or optionally mono- or multiply-substituted by halogen,lower alkyl, lower alkoxy, trifluoromethyl, hydroxyl, carboxylic acid,carboxylic ester, nitro, amino, or phenyl, particularly by halogen,lower alkyl, lower alkoxy, trifluoromethyl, hydroxyl, nitro, amino andphenyl.

[0044] The term “heteroaryl” means a 5- or 6-membered heteroaromaticgroup which contains one or more hetero atoms selected from N, S, and O.

[0045] Preferred generally unreactive R₁ capping groups include methoxy,hydroxyl, or benzyloxy. An especially preferred R₁ capping group ismethoxy. When R₁ is methoxy the aldehydes and related compounds aresometimes referred to herein as “mPEG” compounds, wherein the “m” standsfor methoxy.

[0046] If the R₁ capping group is generally reactive with other chemicalmoieties, then R₁ is a functional group capable of reacting with someother functional group, such as an amine and/or sulfhydryl in a peptideand/or protein. In such a case, R₁ may be a functional group that iscapable of reacting readily with electrophilic or nucleophilic groups onother molecules, in contrast to those groups that require strongcatalysts or highly impractical reaction conditions in order to react.If R₁ is relatively reactive, the polyethylene glycol aldehyde isbifunctional and may therefore covalently bond with two chemicalmoieties.

[0047] Examples of suitable generally reactive R₁ capping groupsinclude: halogen, epoxide, maleimide, orthopyridyl disulfide, tosylate,isocyanate, hydrazine hydrate, cyanuric halide, N-succinimidyloxy,sulfo-N-succinimidyloxy, 1-benzotriazolyloxy, 1-imidazolyloxy,p-nitrophenyloxy, and

[0048] The term “halogen” means fluorine, chlorine, bromine, or iodine.

[0049] A preferred generally reactive R₁ capping group is

[0050] The use of this R₁ group results in a polyethylene glycolaldehyde with aldehyde groups on both ends of the polyethylene glycolaldehyde. And accordingly, the resultant polyethylene glycol aldehydeexhibits binding properties on both ends. It will be appreciated,however, that these bifunctional compounds need not be perfectlysymmetrical, and that the first m, n, and/or p may be the same ordifferent from the second m, n, and/or p in the formula. It ispreferred, however, that the compound be symmetrical, meaning that bothdepicted m's have the same value, both n's have the same value, and bothp's have the same value.

[0051] In the compounds of the present invention X is O or NH.Preferably, X is O. Further, Y is selected from the group consisting of

[0052] wherein Z is a side chain of an amino acid.

[0053] In the present invention, m is from 1 to 17. In a preferredembodiment, m is from 1 to 14. More preferably m is from 1 to 7, andeven more preferably, m is from 1 to 4. Most preferably, m is 1.

[0054] In the case of a Y group with the general structure:

[0055] the Y group exhibits a linkage to the amino acid through apeptide bond.

[0056] Accordingly, this general structure results in specificstructures as simple as:

[0057] when a single glycine is used as the amino acid. When Z is CH₃,then the amino acid is alanine. If Z is CH₂OH, the amino acid is serine.

[0058] Obviously, more complex structures are possible when more anddifferent amino acids are utilized, as can be appreciated from anexamination of the various amino acid structures shown below.Preferably, only one amino acid is used.

[0059] In the present invention, n is from 10 to 10,000. In a preferredembodiment of the present invention n is from 20 to 5,000. Preferably, nis from 50 to 2,500, even more preferably n is from 75 to 1,000. Mostpreferably, n is from 100 to 750.

[0060] In the present invention, p is from 1 to 3. Preferably, p is 3.

[0061] In preferred embodiments, p is 3, R₁ is methoxy, m is 1, and n isfrom 100 to 750; or p is 2, R₁ is methoxy, m is 1, and n is from 100 to750; or p is 1, R₁ is methoxy, m is 1, and n is from 100 to 750.

[0062] The present invention includes, but is not limited to, compoundsof formula I which are compounds of formulas II-VI as follows:

[0063] Preferred R₁ capping moieties are relatively unreactive, withmethoxy, hydroxyl, and benzyloxy preferred.

[0064] Preferred compounds of the present invention fall within Group Aabove.

[0065] Accordingly, the present invention provides a compound of formula(II):

[0066] wherein R₁, m, n, and p are defined as above.

[0067] In a preferred embodiment, R₁ is methoxy, m is 1, and n is from100 to 750. More preferably, p is 3, R₁ is methoxy, m is 1, and n isfrom 100 to 750.

[0068] Another preferred embodiment of the present invention provides abifunctional polyethylene glycol aldehyde compound of formula (VIII):

[0069] wherein m, n, and p are defined as above.

[0070] In a preferred embodiment, R₁ is methoxy, m is 1, and n is from100 to 750. More preferably, p is 3, R₁ is methoxy, m is 1, and n isfrom 100 to 750.

[0071] The present invention also provides a variety of chemicalintermediates which may be converted into the polyethylene glycolaldehyde compounds of the invention described above. These intermediatesinclude compounds of formula (IX):

R₁—(CH₂—CH₂—O)_(n)—CH₂—CH₂—X—Y—NH—(CH₂)_(p)—CH—(OCH₂—CH₃)₂  (IX)

[0072] wherein R₁, X, Y, Z, m, n, and p are defined as above.

[0073] In a preferred embodiment, p is 3, R₁ is methoxy, m is 1, and nis from 100 to 750; or p is 2, R₁ is methoxy, m is 1, and n is from 100to 750; or p is 1, R₁ is methoxy, m is 1, and n is from 100 to 750.

[0074] The present invention further provides intermediate compounds offormula (X):

[0075] wherein R₁, m, n, and p are defined as above.

[0076] In a preferred embodiment, p is 3, R₁ is methoxy, m is 1, and nis from 100 to 750; or p is 2, R₁ is methoxy, m is 1, and n is from 100to 750; or p is 1, R₁ is methoxy, m is 1, and n is from 100 to 750.

[0077] Also provided are intermediate compounds of formula (XI):

[0078] wherein each m, n, and p is the same or different and is definedas above.

[0079] In preferred embodiments, p is 3, R₁ is methoxy, m is 1, and n isfrom 100 to 750; or p is 2, R₁is methoxy, m is 1, and n is from 100 to750; or p is 1, R₁ is methoxy, m is 1, and n is from 100 to 750.

[0080] The compounds of the present invention may be produced by anysuitable method, using known reagents and methods. However, the presentinvention provides a specific method of making a polyethylene glycolaldehyde comprising hydrolyzing a compound of formula (IX):

R₁—(CH₂—CH₂—O)_(n)—CH₂CH₂—X—Y—NH—(CH₂)_(p)—CH—(OCH₂—CH₃)₂  (IX)

[0081] to produce a polyethylene glycol aldehyde of formula (I):

R₁—(CH₂CH₂O)_(n)—CH₂CH₂—X—Y—NH—(CH₂)_(p)—CHO  (I)

[0082] wherein R₁, X, Y, Z, m, n, and p are defined as above.Preferably, the hydrolysis is acid catalyzed. Suitable catalytic acidsinclude: trifluoroacetic acid, hydrochloric acid, phosphoric acid,sulfuric acid, and nitric acid. Preferably, the acid is trifluoroaceticacid.

[0083] In preferred embodiments, p is 3, R₁ is methoxy, m is 1, and n isfrom 100 to 750; or p is 2, R₁ is methoxy, m is 1, and n is from 100 to750; or p is 1, R₁ is methoxy, m is 1, and n is from 100 to 750.

[0084] The polyethylene glycol aldehyde compounds of formula (II) mayalso be produced by any suitable method. By way of example, however,polyethylene glycol aldehydes of formula (II) may be produced asfollows: First, the polyethylene glycol is dried. Second, thepolyethylene glycol is reacted with a halogenated derivative of aceticacid. Hydrolyzing the resulting reaction mixture results in a PEGcarboxylic acid. Alternatively, the product PEG carboxylic acid may alsobe derived from direct oxidation of the PEG, after the drying step.Next, the PEG carboxylic acid is then treated with an amine derivativeof diethyl acetal to produce a PEG acetal amine, which is reacted with ahalogenated carboxylic acid to produce a polyethylene glycol aldehyde offormula. The polyethylene glycol aldehyde product is then collected andpurified.

[0085] The polyethylene glycol aldehyde product may be collected andpurified in any suitable manner. By way of example, the polyethyleneglycol aldehyde product may be extracted with dichloromethane. Theorganic layer is dried over sodium sulfate, filtered, concentrated, andprecipitated with diethyl ether. The product, PEG aldehyde, is collectedby filtration and dried under vacuum.

[0086] The present invention thus provides a method of making apolyethylene glycol aldehyde comprising hydrolyzing a compound offormula (X):

[0087] to produce a polyethylene glycol aldehyde of formula (II):

[0088] wherein R₁, m, n, and p are defined as above.

[0089] The compound of formula (X) may be produced by reacting acompound of formula (XII):

R₁—(CH₂—CH₂—O)_(n)—CH₂CH₂—O—(CH₂)_(m)—COOH  (XII)

[0090] with a compound of formula (XIII):

H₂N—(CH₂)_(p)—CH—(OCH₂—CH₃)₂  (XIII).

[0091] Another method to make PEG acid or PEG carboxylic acid is directoxidation. In this case, oxidizers such as CrO₃ or K₂Cr₂O₇/H₂SO₄, HNO₃in the presence of ammonium vanadate or Jone's reagent (CrO₃ and H₂SO₄),may be used.

[0092] The compound of formula (XII) may be produced by hydrolyzing acompound of formula (XIV):

R₁—O—(CH₂—CH₂—O)_(n)—CH₂CH₂—O—(CH₂)_(m)—COOR₃  (XIV)

[0093] wherein R₃ is a branched or unbranched C₁-C₄ alkyl.

[0094] The compound of formula (XIV) may be produced by reacting acompound of formula (XV):

R₁—(CH₂—CH₂—O)_(n)—CH₂—CH₂—OH  (XV)

[0095] with a compound of formula (XVI):

R₂—(CH₂)_(m)—COOR₃  (XVI)

[0096] wherein R₂ is halogen. Preferably R₂ is bromine or chlorine.Suitable compounds of formula (XVI) include t-butyl bromoacetate, methylbromoacetate, ethyl bromoacetate, t-butyl chloroacetate, methylchloroacetate, and ethyl chloroacetate. Other reagents that can be usedfor this reaction step, i.e., substitutes for formula (XVI) are, e.g.,t-butyl bromoacetate, methyl bromoacetate, ethyl bromoacetate, t-butylchloroacetate, methyl chloroacetate, or ethyl chloroacetate in thepresence of potassium t-butoxide, an alkali metal hydride such as sodiumhydride or potassium naphtalide. Preferably, the compound of formula(XVI) is t-butyl bromoacetate.

[0097] In preferred embodiments, p is 3, R₁ is methoxy, m is 1, and n isfrom 100 to 750; or p is 2, R₁ is methoxy, m is 1, and n is from 100 to750; or p is 1, R₁ is methoxy, m is 1, and n is from 100 to 750.

[0098] Compounds of formulas (III)-(VI) (also identified as Groups B-E,respectively) may likewise be made by any suitable means. By way ofexample, however, the following reaction schemes may be used to producecompounds of formulas (III)-(VI) (Groups B-E).

[0099] As with the polyethylene glycol aldehydes discussed above,bifunctional polyethylene glycol aldehydes may be produced by anysuitable means. The present invention provides a method of making apolyethylene glycol aldehyde comprising hydrolyzing a compound offormula (XVII):

[0100] to produce a polyethylene glycol aldehyde of formula (VIII):

[0101] wherein m, n, and p are defined as above.

[0102] The compound of formula (VI) may be produced by reacting acompound of formula (XVIII):

HOOC—(CH₂)_(m)—O—CH₂CH₂—(CH₂—CH₂—O)_(n)—CH₂CH₂—O—(CH₂)_(m)—COOH  (XVIII)

[0103] with a compound of formula (XIX):

H₂N—(CH₂)_(p)—CH—(OCH₂—CH₃)₂  (XIX).

[0104] The compound of formula (XVIII) may be produced by hydrolyzing acompound of formula (XX):

R₃OOC—(CH₂)_(m)—CH₂CH₂—O—(CH₂—CH₂—O)_(n)—CH₂CH₂—O—(CH₂)_(m)—COOR₃  (XX)

[0105] wherein R₃ is a branched or unbranched C₁-C₄ alkyl.

[0106] The compound of formula (XX) may be produced by reacting acompound of formula (XXI):

HO—CH₂CH₂—(CH₂—CH₂—O)_(n)—CH₂CH₂—OH  (XXI)

[0107] with a compound of formula (XVI):

R₂—(CH₂)_(m)—COOR₃  (XVI)

[0108] wherein R₂ is halogen.

[0109] The polyethylene glycol aldehyde compositions of the presentinvention discussed above may be used to derivatize a variety ofmolecules, including biomolecules, using any suitable methods.

[0110] The PEG aldehyde compounds of the present invention areN-terminus site-specific for the pegylation of peptides and otherbiomolecules. The PEG aldehydes of the present invention form aconjugate with the N-terminus α-amino group of the biomolecule orprotein forming a stable secondary amine linkage between the PEG and thebiomolecule or protein.

[0111] Biomolecules pegylated with PEG aldehydes of the presentinvention show reproducibility in the number and location of PEGattachment, resulting in a purification strategy that is lesscomplicated. This site-specific pegylation can result in a conjugatewhere the pegylation site is far from the site where the biomolecule orthe peptide binds to the cell's receptors, which will allow pegylatedbiomolecules, proteins, or peptides to retain much or all of theirbiological activity. The PEG-aldehydes of the present invention canreact with any biomolecules that contain an alpha (α) amino group.

[0112] Depending on the polyethylene glycol aldehyde selected thepolyethylene glycol may be covalently bonded to a biomolecule at one end(monofunctional polyethylene glycol aldehyde) or at both ends(bifunctional polyethylene glycol aldehyde).

[0113] As stated, the polyethylene glycol aldehydes of the presentinvention may be used for N-terminus site-specific pegylation. Thesite-specific N-terminal linkage results in pegylated polypeptides whichavoid cross-linking and multiple derivatizations of a singlepolypeptide. To produce this site-specific covalent linkage, anysuitable reaction conditions may be used. Generally, the pH of thereaction mixture is sufficiently acidic to activate the α-amino acid ofthe polypeptide to be pegylated. Typically, the pH is about 5.5 to about7.4, preferably about 6.5.

[0114] Accordingly, a method for attaching a polyethylene glycolmolecule to a polypeptide comprising:

[0115] reacting at least one polypeptide of formula (XXII):

NH₂B  (XXII);

[0116] with a polyethylene glycol aldehyde molecule of formula (I):

R₁—(CH₂CH₂O)_(n)—CH₂CH₂—X—Y—NH—(CH₂)_(p)—CHO  (I)

[0117] wherein R₁, X, Y, Z, m, n, and p are defined as above;

[0118] to produce a compound of formula (XXIII):

R₁—(CH₂CH₂O)_(n)—CH₂CH₂—X—Y—NH—(CH₂)_(p)—NHB  (XXIII)

[0119] wherein the polyethylene glycol aldehyde molecule is bonded tothe N-terminal amino group of the polypeptide is provided.

[0120] In preferred embodiments, p is 3, R₁ is methoxy, m is 1, and n isfrom 100 to 750; or p is 2, R₁ is methoxy, m is 1, and n is from 100 to750; or p is 1, R₁ is methoxy, m is 1, and n is from 100 to 750.

[0121] The compounds of formula (XXII) may be any polypeptide, includinginterferon-alpha, interferon-beta, consensus interferon, erythropoietin(EPO), granulocyte colony stimulating factor (GCSF),granulocyte/macrophage colony stimulating factor (GM-CSF), interleukins(including IL-2, IL-10, and IL-12), and colony stimulating factor.

[0122] The compounds of formula (XXII) may also be immunoglobulins, suchas, IgG, IgE, IgM, IgA, IgD, and subclasses thereof, and fragmentsthereof. The term “antibody” or “antibody fragments” refer to polyclonaland monoclonal antibodies, an entire immunoglobulin or antibody or anyfunctional fragment of an immunoglobin molecule which binds to thetarget antigen. Examples of such antibody fragments include Fv (fragmentvariable), single chain Fv, complementary determining regions (CDRs), VL(light chain variable region), VH (heavy chain variable region), Fab(fragment antigen binding), F(ab)2′, and any combination of those or anyother functional group of an immunoglobin peptide capable of binding toa target antigen.

[0123] As stated, the pegylated compound may be prepared in any desiredmanner. Conditions, e.g., pH, should be selected which favor thesite-specific pegylation of α-amino groups.

[0124] Generally, polypeptides may be pegylated with polyethylene glycolcompounds of the invention by adding the compound of formula (XXII), andthe PEG reagent in a molar ratio range of 1:1 to 1:100. The reactionconcentration may then placed in a borate, phosphate, or tri buffer atroom temperature or 4 degrees Celsius for about 0.5 to 24 hours at a pHrange of 5.5 to 9.0. The molar ratio of PEG reagent to peptide/proteinsis generally from 1:1 to 100:1. The concentration of peptide/proteins isgenerally from 1 to 10 mg/ml. The concentration of buffer is usually 10to 500 mM.

[0125] The pegylated compound may be isolated or purified in any desiredmanner. By way of example, the resultant reaction mixture may be dilutedwith an equilibration buffer (20 mM Tris, pH 7.5) and the resultingmixture is then applied on a Q-Sepharose column. After the mixture isapplied on the QA column, it is washed with the equilibration buffereluted with 75 M NaCl; eluted with 200 mM NaCl; eluted with 1M NaCl; andregenerated with 1M HOAC+1M NaCl and 0.5 NaOH. By using reverse phaseHPLC, it is possible to separate and isolate the N-terminal,monopegylated product from other byproducts in the mixture. Eachcollected product can then be confirmed by Matrix Assisted LaserDesorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF).

[0126] In a preferred embodiment of the pegylation method of theinvention, a polypeptide of formula (XXII):

NH₂B  (XXII);

[0127] is reacted with a polyethylene glycol aldehyde molecule offormula (II):

R₁—(CH₂CH₂O)_(n)—CH₂CH₂—O—(CH₂)_(m)—CO—NH—(CH₂)_(p)—CHO  (II)

[0128] wherein R₁, m, n, and p are defined as above; to produce acompound of formula (XXIV):

R₁—(CH₂CH₂O)_(n)—CH₂CH₂—O—(CH₂)_(m)—CO—NH—(CH₂)_(p)—NHB  (XXIV)

[0129] wherein the polyethylene glycol aldehyde molecule is bonded tothe N-terminal amino group of the polypeptide.

[0130] In preferred embodiments, p is 3, R₁ is methoxy, m is 1, and n isfrom 100 to 750; or p is 2, R₁ is methoxy, m is 1, and n is from 100 to750; or p is 1, R₁ is methoxy, m is 1, and n is from 100 to 750.

[0131] Additional illustrations of the use of the compounds of thepresent invention are disclosed in the U.S. Provisional PatentApplications entitled “Pegylated T20 Polypeptide,” U.S. Ser. No.60/398,195, filed Jul. 24, 2002, and “Pegylated T1249 Polypeptide, U.S.Ser. No. 60/439,213 filed Jan. 10, 2003, and Ser. No. 60/398,190 filedJul. 24, 2002, all of which are incorporated herein by reference as ifrecited in full.

[0132] Further provided, is a method for attaching a polyethylene glycolmolecule to a polypeptide comprising:

[0133] reacting a polypeptide of formula (XXII):

NH₂B  (XXII);

[0134] with a polyethylene glycol aldehyde molecule of formula (VIII):

[0135] wherein each m, n, and p is the same or different and is definedas above; to produce a compound of formula (XXV):

[0136] wherein the polyethylene glycol aldehyde molecule is bonded tothe N-terminal amino group of the polypeptides.

[0137] In preferred embodiments, p is 3, m is 1, and n is from 100 to750; or p is 2, m is 1, and n is from 100 to 750; or p is 1, m is 1, andn is from 100 to 750.

[0138] The pegylated polypeptides may be used in any desired manner.Suitably, however, they are used to prepare pharmaceutical compositions,by admixture with a pharmaceutically acceptable excipient. Suchpharmaceutical compositions may be in unit dosage form. They may beinjectable solutions or suspensions, transdermal delivery devices, orany other desired form.

[0139] The following examples are provided to further illustrate thepresent invention. These examples are illustrative only and are notintended to limit the scope of the invention in any way.

EXAMPLES Example 1

[0140] Preparation of PEG Aldehyde Compounds

[0141] Five grams of PEG (molecular weight of 1,000 to 60,000 daltons)in 50 to 100 ml of toluene is azeotropically dried by refluxing for 1 to3 hours, followed by the removal of 20 to 30 mL of toluene. Theresulting solution is cooled to room temperature then potassiumtert-butoxide (1 to 10 molar excess) in 20-50 ml of absolutetert-butanol and 20-50 ml of toluene is added to the PEG solution. Theresulting mixture is then stirred for two hours at room temperatureunder argon.

[0142] Tert-butyl bromoacetate (1 to 10 molar excess) is added to thereaction via syringe and the reaction mixture stirred overnight at roomtemperature under argon gas. Depending on the desired size of the “m”group defined in formula (XVI), tert-butyl bromoacetate can be replacedwith another halogenated derivative of acetic acid, e.g., propionicacid, butyric acid, etc.

[0143] The reaction solution is then condensed by rotary evaporation andthe residue precipitated by the addition of diethyl ether. Theprecipitated product, PEG t-butyl carboxy ester, is filtered off anddried in vacuo.

[0144] PEG t-butyl carboxy ester (4 g) is then dissolved in 50 to 100 mlof 1N sodium hydroxide and the solution stirred at room temperatureovernight. The pH of the mixture is adjusted to 2.5 to 3.0 by additionof 1 to 6N hydrochloric acid, and the mixture extracted withdichloromethane. The organic layer is then dried over sodium sulfate,filtered, concentrated, and precipitated into diethyl ether. Theproduct, PEG-carboxylic acid, is collected by filtration and dried undervacuum.

[0145] The PEG-carboxylic acid (3 g) is then dissolved in anhydrousdichloromethane (20-30 ml) followed by the addition of4-aminobutylraldehyde diethyl acetal (1-5 molar excess),1-hydroxybenzotriazole (1-5 molar excess), and dicyclohexylcarbodiimide(1-5 molar excess). Depending on the desired size of the “p” groupdefined in formula (XIII), 4-aminobutyraldehyde diethyl acetal can bereplaced with another amine derivative of diethyl acetal, e.g.,3-aminopropionaldehyde diethyl acetal or 2-aminoacetalaldehyde diethylacetal.

[0146] The resulting mixture is stirred overnight at room temperatureunder argon gas. The reaction mixture is filtered, concentrated, andprecipitated with a mixture of 2-propanol and diethyl ether (1:1). ThePEG acetal product is dried in vacuo overnight.

[0147] The PEG acetal product is then dissolved in 10-200 ml of 1-90%CF₃COOH, and the solution is stirred at room temperature overnight. ThepH of the mixture is adjusted to 6.0 by addition of 1 N NaOH solution,and sodium chloride (10 wt %) is then added and the pH of the solutionis adjusted to 7.0 by addition of 1 N NaOH. The mixture is thenextracted with dichloromethane. The organic layer is dried over sodiumsulfate, filtered, concentrated, and precipitated with diethyl ether.The product, PEG aldehyde, is collected by filtration and dried undervacuum.

Example 2

[0148] Preparation of mPEG_(10K)-butanoaldehyd

[0149] The following represents a general reaction scheme for preparingmPEG10k-butanoaldehyde of the invention:

[0150] Reaction Scheme for mPEG_(10k)-butanoaldehyde

[0151] First, Carboxymethyl PEG (mPEG) of molecular weight 10,000daltons (30.0 g, 3 mmol) in 300 mL of toluene was azeotropically driedby refluxing for 2 hours, followed by the removal of 100 ml of toluene.The resulting solution was cooled to room temperature then potassiumtert-butoxide (0.68 g, 6 mmol) in 20 ml of absolute tert-butanol and 20ml of toluene was added to the PEG solution (1). The resulting mixturewas stirred for two hours at room temperature under argon.

[0152] Tert-butyl bromoacetate (1.00 mL, 6.75 mmol) was added to thereaction via syringe and the reaction was stirred overnight at roomtemperature under argon. The reaction solution was then condensed byrotary evaporation. The residue was precipitated by addition of diethylether. The precipitated product was filtered off and dried in vacuo.Yield: 28 g. NMR (d₆-DMSO): 1.40 ppm (t, 9H, —CH3); 3.21 ppm (s, —OCH₃);3.50 ppm (s, —O—CH₂CH₂—O—); 3.96 ppm (s, 2H, —O—CH₂—COO—).

[0153] Next, mPEG_(10k) t-butyl carboxymethyl ester (20 g) was dissolvedin 200 mL of 1N sodium hydroxide and the solution was stirred at roomtemperature overnight (2). The pH of the mixture was adjusted to 2.5 byaddition of 6 N hydrochloric acid, and the mixture was extracted withdichloromethane (50 mL, 40 mL, and 30 mL). The organic layer was driedover sodium sulfate, filtered, concentrated, and precipitated withdiethyl ether. The product, mPEG_(10k)-carboxymethyl acid, was collectedby filtration and dried under vacuum. Yield: 18 g. NMR (d₆-DMSO): 3.21ppm (s, —OCH₃); 3.5 ppm (s, —O—CH₂CH₂—O—); 3.99 ppm (s, 2H,—O—CH₂—COOH).

[0154] The mPEG_(10k)-carboxymethyl acid (3 g, 0.3 mmol) was dissolvedin anhydrous dichloromethane (20 mL) followed by the addition of4-aminobutyraldehyde diethyl acetal (50 mg, 0.3 mmol),1-hydroxybenzotriazole (40 mg, 0.3 mmol), and dicyclohexylcarbodiimide(80 mg, 0.39 mmol) (3). The mixture was stirred overnight at roomtemperature under argon. The reaction mixture was filtered,concentrated, and precipitated with a mixture of 2-propanol and diethylether (1:1). The product, mPEG_(10k)-butanoacetal, was dried in vacuoovernight. Yield: 2.7 g. NMR (d₆-DMSO): 1.07-1.12 ppm (t, 6H,(—O—CH₂—CH₃)₂); 1.46 ppm (m, 4H, —NHCH₂CH₂CH₂—CH—); 3.08-3.11 ppm (q,2H, —NHCH₂CH₂CH₂—CH—); 3.21 ppm (s, —OCH₃); 3.5 ppm (s, —O—CH₂CH₂—O—);3.85 ppm (s, 2H, —O—CH₂—CO—NH—); 4.44 ppm (t, 1 H, —NHCH₂CH₂CH₂—CH—);7.67 ppm (—NH—).

[0155] Finally, the mPEG_(10k)-butanoacetal (5 g, 0.5 mmol) wasdissolved in 50 mL of 10% CF₃COOH and the solution was stirred at roomtemperature overnight (4). The pH of the mixture was adjusted to 6.0 byaddition of 1 N NaOH solution, and sodium chloride (10 wt %) was addedand then the pH of the solution was adjusted to 7.0 by addition of 1 NNaOH. The mixture was extracted with dichloromethane. The organic layerwas dried over sodium sulfate, filtered, concentrated, and precipitatedinto diethyl ether. The product, mPEG_(10k)-butanoaldehyde (5), wascollected by filtration and dried under vacuum. Yield: 4.1 g (82%). NMR(d₆-DMSO): 3.21 ppm (s, —OCH₃); 3.5 ppm (s, -0-CH₂CH₂—O); 3.85 ppm (s,2H, —O—CH₂—CO—NH—); 7.67 ppm (—NH—); 9.66 ppm (—CHO—).

Example 3

[0156] Preparation of mPEG_(10k)-acetal Aldehyde

[0157] mPEG_(10k)-acetal aldehyde was prepared by dissolvingmPEG_(10k)-diethyl acetal (1 g, Mol. Wt. 10,000), which was preparedaccording to the procedure in Example 1, in 10 ml of 80% trifluoaceticacid (Aldrich, 99+%). The reaction solution was stirred overnight atroom temperature under argon gas. 1N NaOH was then added dropwise to thereaction solution until a pH of 6.0 was obtained. Next, NaCl (10 wt %)was added to the above solution. The pH was then adjusted to 6.95±0.05by adding 0.1 N NaOH. The solution was then extracted with methylenechloride. The organic layer was then dried over sodium sulfate,filtered, concentrated, and precipitated with diethyl ether. Theproduct, mPEG_(10k)-acetal aldehyde, was collected by filtration anddried under vacuum. Yield: 0.85 g (85%).

Example 4

[0158] Preparation of mPEG_(10k)-propionaldehyde

[0159] mPEG_(10k)-propionaldehyde was prepared by dissolvingmPEG_(10k)-propionacetal (2 g, Mol. Wt. 10,000), which was preparedaccording to the procedure in example 1, in 20 ml of 80% trifluoaceticacid (Aldrich, 99+%). The reaction solution was stirred overnight atroom temperature under argon gas. 1N NaOH was then added dropwise to thereaction solution until a pH of 6.0 was obtained. Next, NaCl (10 wt %)was added to the above solution. The pH was then adjusted to 6.95±0.05by adding 1 N NaOH. The solution was then extracted with methylenechloride. The organic layer was then dried over sodium sulfate,filtered, concentrated, and precipitated with diethyl ether. Theproduct, mPEG_(10k)-propionaldehyde, was collected by filtration anddried under vacuum. Yield: 1.8 g (90%).

Example 5

[0160] Preparation of PEG_(20k)-di-butanoaldehyde

[0161] PEG_(20k)-di-butanoaldehyde was prepared by dissolvingPEG_(20k)-di-butyraldehyde diethyl acetal (3.1 g, Mol. Wt. 20,000),which was prepared according to the procedure in example 1, in 20 ml of80% trifluoacetic acid (Aldrich, 99+%). The reaction solution wasstirred overnight at room temperature under argon gas. 1N NaOH was thenadded dropwise to the reaction solution until a pH of 6.0 was obtained.Next, NaCl (10 wt %) was added to the above solution. The pH was thenadjusted to 6.95±0.05 by adding 0.1 N NaOH. The solution was thenextracted with methylene chloride. The organic layer was then dried oversodium sulfate, filtered, concentrated, and precipitated with diethylether. The product, PEG_(20k)-di-butanoaldehyde, was collected byfiltration and dried under vacuum. Yield: 2.5 g (81%).

Example 6

[0162] Preparation of mPEG_(20k)-butanoaldehyde

[0163] mPEG_(20k)-butanoaldehyde was prepared by dissolvingmPEG_(20k)-butyraldehyde diethyl acetal (3.0 g, Mol. Wt. 20,000), whichwas prepared according to the procedure in Example 1, in 30 ml of 80%trifluoacetic acid (Aldrich, 99+%). The reaction solution was stirredovernight at room temperature under argon gas. 1N NaOH was then addeddropwise to the reaction solution until a pH of 6.0 was obtained. Next,NaCl (10 wt %) was added to the above solution. The pH was then adjustedto 6.95±0.05 by adding 1 N NaOH. The solution was then extracted withmethylene chloride. The organic layer was then dried over sodiumsulfate, filtered, concentrated, and precipitated with diethyl ether.The product, mPEG_(20k)-butanoaldehyde, was collected by filtration anddried under vacuum. Yield: 2.5 g (83.3%).

Example 7

[0164] Preparation of mPEG_(20k)-butanoaldehyde

[0165] mPEG_(20k)-butanoaldehyde was prepared by dissolvingmPEG_(20k)-butyraldehyde diethyl acetal (14.7 g, Mol. Wt. 20,000), whichwas prepared according to the procedure in Example 1, in 200 ml of 10%trifluoacetic acid (Aldrich, 99+%). The reaction solution was stirredovernight at room temperature under argon gas. 1N NaOH was then addeddropwise to the reaction solution until a pH of 6.0 was obtained. Next,NaCl (10 wt %) was added to the above solution. The pH was then adjustedto 6.95±0.05 by adding 0.1 N NaOH. The solution was then extracted withmethylene chloride. The organic layer was then dried over sodiumsulfate, filtered, concentrated, and precipitated with diethyl ether.The product, mPEG_(20k)-butanoaldehyde, was collected by filtration anddried under vacuum. Yield: 13.1 g (89%).

What is claimed is:
 1. A compound of formula (I):R₁—(CH₂CH₂O)_(n)—CH₂CH₂—X—Y—NH—(CH₂)_(p)—CHO  (I) wherein R₁ is acapping group, X is O or NH, Y is selected from the group consisting of

Z is a side chain of an amino acid, m is from 1 to 17, n is from 10 to10,000, and p is from 1 to
 3. 2. A compound according to claim 1,wherein R₁ is selected from the group consisting of halogen, epoxide,maleimide, orthopyridyl disulfide, tosylate, isocyanate, hydrazinehydrate, cyanuric halide, N-succinimidyloxy, sulfo-N-succinimidyloxy,1-benzotriazolyloxy, 1-imidazolyloxy, p-nitrophenyloxy, and


3. A compound according to claim 1, wherein R₁ is selected from thegroup consisting of hydrogen, hydroxy, lower alkyl, lower alkoxy, lowercycloalkyl, lower alkenyl, aryl, and heteroaryl.
 4. A compound accordingto claim 1, wherein R₁ is selected from the group consisting of methoxy,hydroxy, and benzyloxy.
 5. A compound according to claim 2, wherein R₁is


6. A compound according to claim 1 having the formula (III):


7. A compound according to claim 1 having the formula (IV):


8. A compound according to claim 1 having the formula (V):


9. A compound according to claim 1 having the formula (VI):


10. A compound of formula (II):

wherein R₁ is a capping group, m is from 1 to 17, n is from 10 to10,000, and p is from 1 to
 3. 11. A compound according to claim 10,wherein p is
 3. 12. A compound according to claim 11, wherein R₁ isselected from the group consisting of methoxy, hydroxy, and benzyloxy.13. A compound according to claim 11, wherein m is from 1 to
 14. 14. Acompound according to claim 13, wherein m is from 1 to
 7. 15. A compoundaccording to claim 14, wherein m is from 1 to
 4. 16. A compoundaccording to claim 11, wherein n is from 20 to 5,000.
 17. A compoundaccording to claim 16, wherein n is from 50 to 2,500.
 18. A compoundaccording to claim 17, wherein n is from 75 to 1,000.
 19. A compoundaccording to claim 10, wherein p is 3, R₁ is methoxy, m is 1, and n isfrom 100 to
 750. 20. A compound according to claim 10, wherein p is 2.21. A compound according to claim 20, wherein R₁ is selected from thegroup consisting of methoxy, hydroxy, or benzyloxy.
 22. A compoundaccording to claim 20, wherein m is from 1 to
 14. 23. A compoundaccording to claim 22, wherein m is from 1 to
 7. 24. A compoundaccording to claim 23, wherein m is from 1 to
 4. 25. A compoundaccording to claim 20, wherein n is from 20 to 5,000.
 26. A compoundaccording to claim 25, wherein n is from 50 to 2,500.
 27. A compoundaccording to claim 26, wherein n is from 75 to 1,000.
 28. A compoundaccording to claim 10, wherein p is 2, R₁ is methoxy, m is 1, and n isfrom 100 to
 750. 29. A compound according to claim 10, wherein p is 1.30. A compound according to claim 29, wherein R₁ is selected from thegroup consisting of methoxy, hydroxy, or benzyloxy.
 31. A compoundaccording to claim 29, wherein m is from 1 to
 14. 32. A compoundaccording to claim 31, wherein m is from 1 to
 7. 33. A compoundaccording to claim 32, wherein m is from 1 to
 4. 34. A compoundaccording to claim 29, wherein n is from 20 to 5,000.
 35. A compoundaccording to claim 34, wherein n is from 50 to 2,500.
 36. A compoundaccording to claim 35, wherein n is from 75 to 1,000.
 37. A compoundaccording to claim 10, wherein p is 1, R₁ is methoxy, m is 1, and n isfrom 100 to
 750. 38. A compound of formula (VIII):

wherein m is from 1 to 17, n is from 10 to 10,000, and p is from 1 to 3.39. A compound according to claim 38, wherein m is from 1 to
 14. 40. Acompound according to claim 39, wherein m is from 1 to
 7. 41. A compoundaccording to claim 40, wherein m is from 1 to
 4. 42. A compoundaccording to claim 38, wherein n is from 20 to 5,000.
 43. A compoundaccording to claim 42, wherein n is from 50 to 2,500.
 44. A compoundaccording to claim 43, wherein n is from 75 to 1,000.
 45. A compoundaccording to claim 38, wherein p is 3, m is 1 and n is from 100 to 750.46. A compound of formula (IX):R₁—(CH₂—CH₂—O)_(n)—CH₂—CH₂—X—Y—NH—(CH₂)_(p)—CH—(OCH₂—CH₃)₂  (IX) whereinR₁ is a capping group, X is O or NH, Y is selected from the groupconsisting of

Z is a side chain of an amino acid, m is from 1 to 17, n is from 10 to10,000, and p is from 1 to
 3. 47. A compound according to claim 46,wherein R₁ is selected from the group consisting of halogen, epoxide,maleimide, orthopyridyl disulfide, tosylate, isocyanate, hydrazinehydrate, cyanuric halide, N-succinimidyloxy, sulfo-N-succinimidyloxy,1-benzotriazolyloxy, 1-imidazolyloxy, p-nitrophenyloxy, and


48. A compound according to claim 46, wherein R₁ is selected from thegroup consisting of hydrogen, hydroxy, lower alkyl, lower alkoxy, lowercycloalkyl, lower alkenyl, aryl, and heteroaryl.
 49. A compoundaccording to claim 46, wherein R₁ is selected from the group consistingof methoxy, hydroxy, and benzyloxy.
 50. A compound according to claim46, wherein R₁ is


51. A compound according to claim 46, wherein m is from 1 to
 14. 52. Acompound according to claim 51, wherein m is from 1 to
 7. 53. A compoundaccording to claim 52, wherein m is from 1 to
 4. 54. A compoundaccording to claim 46, wherein n is from 20 to 5,000.
 55. A compoundaccording to claim 54, wherein n is from 50 to 2,500.
 56. A compoundaccording to claim 55, wherein n is from 75 to 1,000.
 57. A compoundaccording to claim 46, wherein R₁ is methoxy, p is 3, m is 1, and n isfrom 100 to
 750. 58. A compound of formula (X):

wherein R₁ is a capping group, m is from 1 to 17, n is from 10 to10,000, and p is from 1 to
 3. 59. A compound according to claim 58,wherein R₁ is selected from the group consisting of halogen, epoxide,maleimide, orthopyridyl disulfide, tosylate, isocyanate, hydrazinehydrate, cyanuric halide, N-succinimidyloxy, sulfo-N-succinimidyloxy,1-benzotriazolyloxy, 1-imidazolyloxy, p-nitrophenyloxy, and


60. A compound according to claim 58, wherein R₁ is selected from thegroup consisting of hydrogen, hydroxy, lower alkyl, lower alkoxy, lowercycloalkyl, lower alkenyl, aryl, and heteroaryl.
 61. A compoundaccording to claim 58, wherein R₁ is selected from the group consistingof methoxy, hydroxy, and benzyloxy.
 62. A compound according to claim58, wherein R₁ is


63. A compound according to claim 62, wherein m is from 1 to
 14. 64. Acompound according to claim 63, wherein m is from 1 to
 7. 65. A compoundaccording to claim 64, wherein m is from 1 to
 4. 66. A compoundaccording to claim 58, wherein n is from 20 to 5,000.
 67. A compoundaccording to claim 66, wherein n is from 50 to 2,500.
 68. A compoundaccording to claim 67, wherein n is from 75 to 1,000.
 69. A compoundaccording to claim 58, wherein R₁ is methoxy, p is 3, m is 1, and n isfrom 100 to
 750. 70. A compound of formula (XI):

wherein m is from 1 to 17, n is from 10 to 10,000, and p is from 1 to 3.71. A compound according to claim 70, wherein m is from 1 to
 14. 72. Acompound according to claim 71, wherein m is from 1 to
 7. 73. A compoundaccording to claim 72, wherein m is from 1 to
 4. 74. A compoundaccording to claim 70, wherein n is from 20 to 5,000.
 75. A compoundaccording to claim 74, wherein n is from 50 to 2,500.
 76. A compoundaccording to claim 75, wherein n is from 75 to 1,000.
 77. A compoundaccording to claim 70, wherein p is 3, m is 1 and n is from 100 to 750.78. A method of making a polyethylene glycol aldehyde comprisinghydrolyzing a compound of formula (IX):R₁—(CH₂—CH₂—O)_(n)—CH₂CH₂—X—Y—NH—(CH₂)_(p)—CH—(OCH₂—CH₃)₂  (IX) toproduce a polyethylene glycol aldehyde of formula (l):R₁—(CH₂CH₂O)_(n)—CH₂CH₂—X—Y—NH—(CH₂)_(p)—CHO  (I) wherein R₁ is acapping group, X is O or NH, Y is selected from the group consisting of

Z is a side chain of an amino acid, m is from 1 to 17, n is from 10 to10,000, and p is from 1 to
 3. 79. A method of making a polyethyleneglycol aldehyde comprising hydrolyzing a compound of formula (X):

to produce a polyethylene glycol aldehyde of formula (II):

wherein R₁ is a capping group, m is from 1 to 17, n is from 10 to10,000, and p is from 1 to
 3. 80. A method according to claim 79 whereinthe compound of formula (X) is produced by reacting a compound offormula (XII): R₁—(CH₂—CH₂—O)_(n)—CH₂CH₂—O—(CH₂)_(m)—COOH  (XII) with acompound of formula (XII): H₂N—(CH₂)_(p)—CH—(OCH₂—CH₃)₂  (XIII).
 81. Amethod according to claim 80 wherein the compound of formula (XII) isproduced by hydrolyzing a compound of formula (XIV):R₁—O—(CH₂—CH₂—O)_(n)—CH₂CH₂—O—(CH₂)_(m)—COOR₃  (XIV) wherein R₃ is abranched or unbranched C₁-C₄ alkyl.
 82. A method according to claim 81wherein the compound of formula (XIV) is produced by reacting a compoundof formula (XV): R₁—(CH₂—CH₂—O)_(n)—CH₂CH₂—OH  (XV) with a compound offormula (XVI): R₂—(CH₂)_(m)—COOR₃  (XVI) wherein R₂ is halogen.
 83. Amethod of making a polyethylene glycol aldehyde comprising hydrolyzing acompound of formula (XVII):

to produce a polyethylene glycol of formula (VIII):

wherein m is from 1 to 17, n is from 10 to 10,000, and p is from 1 to 3.84. A method according to claim 83 wherein the compound of formula(VIII) is produced by reacting a compound of formula (XVIII):HOOC—(CH₂)_(m)—O—CH₂CH₂—(CH₂—CH₂—O)_(n)—CH₂CH₂—O—(CH₂)_(m)—COOH  (XVIII)with a compound of formula (XIX): H₂N—(CH₂)_(p)—CH—(OCH₂—CH₃)₂  (XIX).85. A method according to claim 84 wherein the compound of formula(XVIII) is produced by hydrolyzing a compound of formula (XX):R₃OOC—(CH₂)_(m)—CH₂CH₂—O—(CH₂—CH₂—O)_(n)—CH₂CH₂—O—(CH₂)_(m)—COOR₃  (XX)wherein R₃ is a branched or unbranched C₁-C₄ alkyl.
 86. A methodaccording to claim 85 wherein the compound of formula (XX) is producedby reacting a compound of formula (XXI):HO—CH₂CH₂—(CH₂—CH₂—O)_(n)—CH₂CH₂—OH  (XXI) with a compound of formula(XVI): R₂—(CH₂)_(m)—COOR₃  (XVI) wherein R₂ is halogen.